Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.     

The continuing story of endoribonuclease III

Endoribonuclease III (RNase III) plays an important role in the processing of rRNA and mRNAs. It is timely to summarize the most relevant insights obtained during the last years stemming from RNase III. With this aim, the present mini-review provides a wealth of new information focusing on the distribution and architecture of RNase III, substrate recognition, cleavage mechanisms and regulation of gene expression.     

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected.     

A Systematic Critical Appraisal of Clinical Practice Guidelines in Juvenile Idiopathic Arthritis Using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) Instrument

Objectives

The objectives of this review are to: 1) appraise the methodological quality of clinical practice guidelines (CPGs) in juvenile idiopathic arthritis (JIA) providing pharmacological and/or non-pharmacological intervention recommendations, and 2) summarize the recommendations provided by the included CPGs and compare them where possible.

Methods

A systematic search was performed. Three trained appraisers independently evaluated the methodological quality of the CPGs using a validated and reliable instrument, the Appraisal of Guidelines in Research and Evaluation II. Six domains were considered: 1) score and purpose; 2) stakeholder involvement; 3) rigor of development; 4) clarity of presentation; 5) applicability; and 6) editorial independence. The domains consist of a total of 23 items each scored on a 7-point scale. High quality CPGs were identified if they had a domain score above 60% in rigor of development, and two other domains.

Results

Of the three included CPGs, the Royal Australian College of General Practitioners (RACGP) and American College of Rheumatology (ACR) CPGs were considered to be of high quality, but the German Society for Pediatric Rheumatology was of lower quality. Domains one to four had high domain scores across the guidelines (mean (standard deviation)): 72.76 (13.80); 66.67 (9.81); 64.67 (7.77); and 87.00 (9.64), respectively. Lower scores were obtained for applicability (14.00 (5.57)) and editorial independence (43.44 (7.02)). Recommendations varied across CPGs due to differences in context, target audience (general practitioners, rheumatologists, and other multidisciplinary healthcare professionals) and patients’ disease presentations. Despite this variability, progression of pharmacological treatment did not conflict between CPGs. Recommendations for non-pharmacological interventions were vague and the interventions considered varied between CPGs.

Conclusions

Overall, recommendations were based on a paucity of evidence and weak study designs. Further research is needed on interventions in JIA, as well as higher quality CPGs to facilitate implementation of the best evidence-based recommendations in clinical practice.     

5'-to-3' exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5' stability of mRNA

Although the primary mechanism of eukaryotic messenger RNA decay is exoribonucleolytic degradation in the 5'-to-3' orientation, it has been widely accepted that Bacteria can only degrade RNAs with the opposite polarity, i.e. 3' to 5'. Here we show that maturation of the 5' side of Bacillus subtilis 16S ribosomal RNA occurs via a 5'-to-3' exonucleolytic pathway, catalyzed by the widely distributed essential ribonuclease RNase J1. The presence of a 5'-to-3' exoribonuclease activity in B. subtilis suggested an explanation for the phenomenon whereby mRNAs in this organism are stabilized for great distances downstream of "roadblocks" such as stalled ribosomes or stable secondary structures, whereas upstream sequences are never detected. We show that a 30S ribosomal subunit bound to a Shine Dalgarno-like element (Stab-SD) in the cryIIIA mRNA blocks exonucleolytic progression of RNase J1, accounting for the stabilizing effect of this element in vivo.     

Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences

We show that structural type A and B bacterial ribonuclease P (RNase P) RNAs can fully replace each other in vivo despite the many reported differences in their biogenesis, biochemical/biophysical properties and enzyme function in vitro. Our findings suggest that many of the reported idiosyncrasies of type A and B enzymes either do not reflect the in vivo situation or are not crucial for RNase P function in vivo, at least under standard growth conditions. The discrimination of mature tRNA by RNase P, so far thought to prevent product inhibition of the enzyme in the presence of a large cellular excess of mature tRNA relative to the precursor form, is apparently not crucial for RNase P function in vivo.